Project Summary/Abstract (Mark Nesbit) The superfamily of radical S-adenosylmethionine (SAM) enzymes (RSEs) are responsible for catalyzing a wide variety of unusual and difficult chemical reactions which are critical for the survival of living organisms from bacteria to humans. RSEs are identified by binding of a redox active [4Fe-4S] cluster through three cysteine residues (typically from a CX3CX2C sequence). The initial step in RSE catalyzed reactions where SAM binds to the [4Fe-4S] cluster and is reduced to generate Ado? (5?-deoxyadenosyl radical) appears to be common to all RSEs. However, despite having many structural similarities and sharing a common initiation step RSEs are able to catalyze a wide variety of chemical transformations and are involved in peptide modification, metalloprotein cluster assembly and cofactor biosynthesis. Two RSEs involved in peptide modification are PqqE and MftC. PqqE catalyzes a C-C bond forming step in the biosynthesis of the redox cofactor PQQ. MftC catalyzes the oxidative decarboxylation of the C-terminus tyrosine residue in the MftA peptide as a part of the biosynthesis of the proposed redox cofactor mycofactocin. Studying the mechanisms by which these genetically related enzymes are able to catalyze vastly different chemical reactions will provide valuable insight into the varied functionality of RSEs in nature. The planned investigation will interrogate the mechanism of the C-C bond forming step in PQQ biosynthesis catalyzed by the radical SAM enzyme PqqE and the oxidative decarboxylation of a C-terminus tyrosine residue catalyzed by MftC. Continuous wave EPR, and pulsed methods such as ENDOR will be able to intimately probe the nature of potential radical organic and organometallic intermediates. The use of non-natural amino acids designed to stabilize potential radical intermediates and incorporated into analogs of the peptide substrates will assist in these experiments. Additional spectroscopic methods such as rapid freeze-quench 57Fe Mssbauer spectroscopy will provide data which complements the EPR studies allowing for observation of all Fe nuclei in a sample under turnover conditions. The data produced by these experiments will help to further the mechanistic understanding of the diverse array of biological reactions catalyzed by radical SAM enzymes and provide additional characterization of physical and electronic structures of the PqqE and MftC under catalytically relevant conditions. Additionally, these methods may be used to study other RSEs and will provide a strong spectroscopic foundation of knowledge about the mechanisms by which this diverse family of enzymes accomplishes the unique biological roles they have adapted to fill.